Phospho-SLP-76 (Ser376) cellular kit
All-in-one kit for robust detection of Phospho-SLP-76
The Total PLCg1 kit is designed to monitor the expression level of cellular PLCg1 (Phospholipase C), and can be used as a normalization assay for the Phospho-PLCg1 (Tyr783) kit.
This HTRF cell-based assay conveniently and accurately detects Phospholipase C gamma 1 (PLCgamma 1) protein.
PLCgamma1 is a phosphoinositide-specific phospholipase that generates second messengers and plays a crucial role in signal transduction. Upon cell stimulation, PLCgamma 1 interacts with tyrosine kinase receptors and others, like T cell receptors, leading to the activation by phosphorylation at Ty783.
PLCgamma 1 is a potential target in the context of a number of diseases. For example, frequent mutations in the PLCG1 gene have been reported in angiosarcoma and T-cell lymphomas and its activation has also been implicated in the spreading of breast cancer.
The Total PLCg1 assay quantifies the expression level of PLCg1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-PLCg1 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of PLCg1 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein's expression under a no-wash assay format.
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Total-PLCg1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Detection of Total PLCg1 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
The human Jurkat T cell line was seeded in a half area 96-well culture-treated plate at 200,000 cells/well in 25 µL of complete culture medium. After a 3 hour incubation at 37 °C, 5% CO2, cells were stimulated with 5 µL of increasing concentrations of an CD3 antibody for 2 minutes, and then lyzed with 10 µL of supplemented lysis buffer #3 (4X) for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Phospho-PLCg1 (Tyr783) or Total-PLCg1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.
The anti-CD3 antibody induced a dose-dependent increase in PLCg1 phosphorylation at Tyr783 without changing the expression level of the protein, demonstrating the activation of the TCR complex at the cell surface.
The human A431 cell line was seeded in a 96-well culture-treated plate at 50,000 cells/well in 50 µL of complete culture medium. After overnight incubation at 37 °C, 5% CO2, cells were stimulated with 50 µL of increasing concentrations of EGF for 5 minutes. After 30 minutes of lysis incubation, phosphorylated and Total PLCg1 were measured using the two-plate assay protocol. The HTRF signal was recorded after an overnight incubation.
The EGF induced a dose-dependent increase in PLCg1 phosphorylation at Tyr783 without changing the expression level of the protein, demonstrating the activation of tyrosine kinase receptor EGFR at the cell surface.
The mouse NIH-3T3 cell line was seeded in a 96-well culture-treated plate at 100,000 cells/well in 50 µL of complete culture medium. After overnight incubation at 37 °C, 5% CO2, cells were stimulated with 50 µL of increasing concentrations of PDGFR-BB for 30 minutes.
After 30 minutes of lysis incubation, phosphorylated and Total PLCg1 were measured using the two-plate assay protocol. The HTRF signal was recorded after an overnight incubation.
The PDGFR-BB induced a dose-dependent increase in PLCg1 phosphorylation at Tyr783 without changing the expression level of the protein, demonstrating the activation of tyrosine kinase receptor PDGFR at the cell surface.
The human Jurkat T cell line was cultured in a T175 flask in complete culture medium for 48h at 37 °C, 5% CO2. After centrifugation, pelleted cells were stimulated with 5 µg/mL of an anti-CD3 antibody for 2 minutes. Then lysis buffer #3 (2X) was added for 30 minutes at RT under gentle shaking.
Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF total-PLCg1 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.
Using the HTRF total-PLCg1 assay, 1,000 cells/well were enough to detect a significant signal, while 8,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF phospho-PLCg1 assay was 8 times more sensitive than the Western Blot technique.
PLCg1 (Phospholipase C gamma 1) is a cytoplasmic tyrosine kinase which plays a crucial role in the signal transduction of receptor or non-receptor tyrosine kinase.
Upon the stimulation of T cells, Src family proteins phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) on the T-cell antigen receptor (TCR). The phosphorylated ITAMs recruit ZAP-70 and SLP-76 which bind to PLCg1, leading to the activation by phosphorylation of its Tyr783 catalytic domain.
Alternatively, the activation of receptor tyosine kinases triggers PLCg1 membrane recruitment through the binding of its N-terminal SH2 domain, which induces PLCg1 phosphorylation at Ty 783.
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